The adaptive T cell immune response is predicated upon the ability to discriminate self from non-self. This functionality arises from intrathymic selection that eliminate thymocytes bearing autoreactive T cell receptor (TCR) specificities while fostering development of thymocytes with useful TCRs. Repertoire formation is dependent on migration of thymocytes at discrete developmental stages through a gauntlet of thymic epithelial cell (TEC) presented self-peptide-MHC complexes enabling appropriate negative (apoptosis) and positive (survival) TCR selection processes. At the double positive (DP) stage, however, thymocytes exhibit migratory properties not easily explained by responses to simple chemokine gradients, suggesting the existence of additional guidance cues. We have shown that regulated surface expression of plexinD1 on DP thymocytes exerts control over their migration in the cortical to medullary direction during selection. By creating Plxnd1 conditional knockout (CKO) mice on a TCR transgenic background, we now show that aberrant cortical location of DP thymocytes following loss of Plxnd1 severely impacts negative selection. How plexinD1 exerts this control in molecular terms is the focus of the current proposal. We demonstrate that plexinD1 holds b1- integrins in an active state on DP thymocytes, allowing adherence to TEC-expressed VCAM-1 and laminin. The interaction of plexinD1 with its soluble medullary ligand, sema3E, releases adhesion, while that of Plxnd1 CKO DP thymocytes is constitutively defective. Strikingly, adhesion and its regulation by sema3E is dynamic, only being manifest under force as occurs upon cell migration. Interrogation of the individual a4b1-VCAM-1 bonds by biomembrane force probe (BFP) indicates that these are catch bonds, and that the sema3E interaction with plexinD1 releases the integrin catch bonds to attenuate dynamic adhesion. Our results represent the first identification of catch bond regulation in any physiological system. In Aim 1 we will use high resolution dSTORM image analysis and BFP techniques to characterize the relationship of plexinD1 to b1- as well as b2-integrins in nanoscale adhesion patches influencing avidity. Additionally, we shall characterize conformation of individual integrin molecules and their functional bonds with specific ligands. In Aim 2 we will identify the proteins interacting with plexinD1 including those interactome components regulating GTPases (such as GAPs and GEFs that mediate extensive crosstalk with integrins) and those stabilizing integrin interactions with the actin cytoskeleton. The docking sites on plexinD1 for these protein interactions will be determined. In Aim 3 we will use viable thymic slice assays to probe plexinD1 regulation of DP thymocyte integrins during migration, and then extend these results to in vivo analysis of negative selection in Plxnd1 CKO and germline Sema3e-/- mice leading to autoimmunity. These results will lay a new foundation for control of integrin adhesion during thymocyte migration and differentiation. Our findings have implications for regulation of integrin function generally, including in axon pathfinding, angiogenesis and tumor metastasis.